Field-deployable molecular workflow for detection, resistance screening, and genotyping of Staphylococcus aureus in bovine mastitis.

Field-deployable LAMP assays enable rapid S. aureus detection and resistance screening in mastitis diagnostics.

This study developed and validated a field-deployable molecular workflow for rapid detection and characterization of Staphylococcus aureus in bovine mastitis. The integrated approach combines simplified DNA extraction with two LAMP assays: one targeting the nuc gene for S. aureus detection (89.5% sensitivity, 100% specificity versus PCR) and another targeting mecA for methicillin-resistance identification. The workflow includes spa gene genotyping using PCR with high-resolution melt curve analysis supported by a genotype confidence percentage mathematical model, validated by DNA sequencing. Testing on 14 culture-confirmed S. aureus milk samples identified nine distinct spa types, demonstrating substantial genetic diversity. The GCP-based model provided reproducible and objective spa type assignment even with subtle melt curve variations. This cost-effective, point-of-care diagnostic solution is particularly applicable to resource-limited and remote farm settings, enabling early detection and antimicrobial resistance screening with subsequent laboratory-based strain characterization.

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